Genome – and HLA-wide scanning and validation for cytotoxic CD8 T cell responses against Mycobacterium tuberculosis EU6-FRAME PROJECT Sheila Tuyet Tang, Ph.D stud. Biologist

2. Vaccine4TB I) peptide prediction Human cytotoxic CD8 T cell responses against novel TB epitopes II) Human cytotoxic CD8 T cell responses against novel TB epitopes Outline 1. Introduction to Tuberculosis (TB)

Introduction

l Introduction - Pandemic Burden ranking 1.IndiaIndia 2.ChinaChina 3.IndonesiaIndonesia 4.NigeriaNigeria 5.South AfricaSouth Africa 6.BangladeshBangladesh 7.PakistanPakistan 8.EthiopiaEthiopia 9.PhilippinesPhilippines 10.KenyaKenya 11.Democratic Republic of CongoDemocratic Republic of Congo 12.Russian FederationRussian Federation 13.Viet NamViet Nam 14.TanzaniaTanzania 15.UgandaUganda 16.BrazilBrazil 17.AfghanistanAfghanistan 18.ThailandThailand 19.MozambiqueMozambique 20.ZimbabweZimbabwe 21.MyanmarMyanmar 22.CambodiaCambodia

What is tuberculosis?  Tuberculosis disease (TB) is caused by a bacterium Mycobacterium tuberculosis (Mtb, also known as tubercle bacillus)  Eradication of tuberculosis is difficult since Mtb is able to remain in the host for a long time as latent or chronic state  Bacille Calmette Guérin (BCG), the current vaccine prevent the spread of TB but do not give full protection against pulmonary TB in adult( the initial infection)  BCG is given to infant in endemic areas  Thorough immunological knowledge of BCG and Mtb are lacking and necessary for the development of a more efficacious TB vaccine

Tuberculosis: Transmission Exposure/Infection 10% Clearance 70% TB Infection (2 bill, ~ 9 mill/yr) Primary infection Death ~2 mill 30% Latent TB 90% Reactivation 5-15% First 2yrs highest chance of developing TB disease Treatment with several drugs for 6 months or more can cure more than 95% of patients If not treated 60 % dies

Cellular immune response Tubercle bacilli enter aveoli Infect* macrophages Within few weeks Th1 immune response CD4+/CD8 + T cells Recruit to lung Cytokines: IL-2, TNFa and IFN-y Tubercle bacilli MACROPHAGE Lysosome +TB ER TB peptide TCR CD8 T cells IFN-g

 Granulomas prevent spread of infection by confining bacteria within a compact collection of several types of immune cells and activated macrophages  Role of these cells: specific ways to isolate inhibit the replication of, and destroy the bacteria Cellular immune response Bacilli engulfed by macrophages Replicate within the macrophages 2-3 weeks before spreading throughout the body 95% contain the bacteria in macrophages But due to Mtb. complex waxy cel wall the bacteria are protected inside the macrophages

2. Vaccine4TB I) peptide prediction Human cytotoxic CD8 T cell responses against novel TB epitopes II) Human cytotoxic CD8 T cell responses against novel TB epitopes Outline 1. Introduction to Tuberculosis (TB)

Vaccine4TB: Peptide prediction

Bioinformatics strategy 1.Read a GenBank file. Extract the proteins from the file 2.Predict binding to HLA supertypes A2 (A0201), A3 (A0301), B7 (B0702) (coverage approx. 80% of the worlds population) 3. Predict proteasome cleavage and TAP binding 4. Calculate combined score using the method of Larsen et al., have been used to predict and select potential epitopes based on a number of different criteria

TBVAC epitopes(14) Best predicted epitopes in the entire proteom(67) Proteins with CD8 epitopes(25) Proteins from vaccine trials (Michel Klein WP3) Selected in proteins - previously described by other groups to have CTL epitopes (Michel Klein WP3) Selected as the best predicted epitopes In the entire Mtb proteome Selection criteria for potential vaccine candidates….(1) 3 epitopes/protein used in vaccine trials (21) Additional epitopes from proteins with CD8 epitopes (43)

Conserved epitopes (69) Selected as the most conserved epitopes with ANN prediction >0.42(<500nM) binding affinity Conservation defined from multiple alignment that have the 9mer epitope: LAGS/Dos regulon sequences (71) Selected as best predicted epitopes in the LAGS/Dos regulon (Michel Klein Fatima Kazi - WP3) T cell epitopes in proteins with B cell epitopes (60) These proteins found by Ugur Sahims group WP4 Selection criteria for potential vaccine candidates….(2)

New set of peptides selected ( after meeting in Leiden) Exp verified secreted epitopes (67) Predicted secreted epitopes (68) Selected as best predicted from TubercuList secreted epitopes (67) proteins containing a predicted signal peptide or predicted to be secreted by other pathways

TBVAC epitopes (14)

3 epitopes /protein used in vaccine trials (21)

Continue.... LAGS/Dos regulon sequences (71) from 13 proteins

Summary 505 peptides of the 800 to be selected in this project have been chosen – binding affinity have been measured and received in Leiden What next ? new selection of the remaining 295 peptides have recently been made in Mainz - Diagnostic peptides - New antigens dicovered by Ganymed - More LAGS

Verification of prediction - Wet lab experiments predictions

2. Vaccine4TB I) peptide prediction Human cytotoxic CD8 T cell responses against novel TB epitopes II) Human cytotoxic CD8 T cell responses against novel TB epitopes Outline 1. Introduction to Tuberculosis (TB)

Vaccines4TB Genome- and HLA-wide scanning and validation of cytotoxc CD8 T cell responses against Mycobacterium tuberculosis WP3 Human cytotoxic CD8 T cell responses against novel TB epitopes Fatima Kazi, Pascale van Weeren, Corine Prins, Tom Ottenhoff, Michèl Klein Department of Immunohematology and Blood Transfusion Leiden University Medical Center Leiden, The Netherlands

Establishment of blood panel for screening peptides Design of assay for testing immunogenicity of peptides Analysis of CD8 response to peptides Identification of novel CD8 T cell epitopes

41 Buffy coats – more coming up Approx 50% PPD+ (purified protein derivative cell culture extract used as the classical Tubeculin/Mantoux test) non-BCG vaccinated A2+, A3+, B7+ (full HLA-typing details) IFN-  ELISA testing for PPD response 6 day assay > 100 pg/ml = PPD+ Blood panel

PBMC (Peripheral blood mononuclear cells) CFSE labelled FACs (cfse /CD56/CD3/CD8) 7 days Acquire 10,000 events in live CD8 gate Assay for peptide screening 1.5x10 5 cells/well + 10ug/ml peptide R1=gate on lymphocytes FSC SSC CD8 SSC + R2=gate on CD8 T cells

Analysing peptide screening R1=gate on lymphocytes (PBMCs) CD3 CD8 CFSE CD8 SSC FSC 1.33% 9.96% 74.7% 9.73% medium peptide PPD PHA

Blood panel 21 donors tested (cfse) PPD+ = 9 donors (43%) PPD- = 12 donors 7/21 (33%) donors responded to Mycobacteria specific Ags

Epitope Prediction TBVAC epitopes(14) Proteins with CD8 epitopes(25) Proteins from vaccine trials (Michel Klein WP3) Selected in proteins - previously described by other groups to have CTL epitopes (Michel Klein WP3) 3 epitopes/protein used in vaccine trials (21) Additional epitopes from proteins with CD8 epitopes (43)

Peptides TBVAC peptides: Ag85A/B, ESAT6, PPE, HBHA TB-CD8 peptides: Mycobacteria tuberculosis H37Rv strain

CD8 T cell proliferation to A2 motif bearing peptides

CD8 T cell proliferation to A3 motif bearing peptides

A3-peptides Binding versus peptide immunogenicity 3/5 donor recognition

CD8 T cell proliferation to B7 motif bearing peptides

B7-peptides Binding versus peptide immunogenicity

PPD-ve individuals do not respond to peptides PPD responses < 1% cfse+ve No responses to peptides A2 donorsA3 donors B7 donors

Peptides Recognised by CD8 T cells A2 peptides A3 peptides B7 peptides 4/11 (36%) 9/13 (70%) 6/14 (43%)

SUMMARY 19/38 predicted peptides induced a CD8 proliferative response The frequency of proliferating CD8 T cell response to peptides varied between individuals Heterogenous response to peptides For A3-peptide responses, 3/5 donors recognised the same peptide: QINELHHSK (CD8-#108-76), suggesting it may be immunodominant peptide

SUMMARY 19/38 peptides induced a CD8 proliferative response The frequency of proliferating CD8 T cell response to peptides varied between individuals Heterogenous response to peptides For A3-peptide responses, 3/5 donors recognised the same peptide QINELHHSK (CD8-#10876), suggesting it may be immunodominant peptide

Best predicted epitopes in the entire proteom(67) Selected as the best predicted epitopes In the entire Mtb proteome Conserved epitopes (69) Screening of new set of peptides Conservation defined from multiple alignment that have the 9mer epitope: Selected as the most conserved epitopes with ANN prediction >0.42(<500nM) binding affinity Visiting Ph.D student

Donor 46(B7) PPD+ 0.69% 0.61% % % 4.47% 1.74% 0.57%3.37% ppd+ in ELISA CD8 CFSE

Donor 29(HLA-B7) PPD+ CFSE 16.50% 6.33% 92.45%o.19% 1.01% 85.23% CD8

Preliminary results: 5/23 peptides B7 ~22% proliferative CD8+Tcell response Concerved hypothetical protein SubCell: Cytoplamic ProFun: Structural protein involved in energy metabolism

Preliminary summery For Best predicted epitopes in the entire proteom(67) peptide set:  No proliferative CD8+ T cell response were observed for A3 peptides from the set of peptide from : Best predicted epitopes in the entire proteom(67)  For the A2 responses => un-going experiments  22% of the B7 peptides were recognized in five of the donors  Two of the B7 peptides were recognized in >1 donors. RPAIVVPAF (#11672) in 3 and RPAPATGAL (#11673) in two of the donors

Preliminary summery For conserved peptide set(69):  Just initiated the screening last week – finished proliferation assay for two donors:# 49 and 53 (HLA-A3/B7)  Feasible to finish before the end of my study-exchange at LUMC, Leiden, Netherlands

FUTURE WORK and MILESTONES Assays for immunological monitoring of cytotoxic CD8 T cells (6 months) CFSE assay Detected CD8 T cell responses to predicted epitopes Cytokine production (on-going) Increase to 10 donor per supertype group CD8 T cell epitopes which can elicit HLA class-I restricted cytotoxic T cell responses with potential anti-microbial activity towards M. tuberculosis (6-8 months) CD8 T cell lines/clones Chromium release assay /blocking Abs Recognise endogenous peptides Granzyme, CD69/CD40L

FUTURE WORK and MILESTONES Understanding the repertoire of CD8 T cell responses induced during (i) natural infection with M. tuberculosis and (ii) BCG vaccination in a cross sectional and retrospective study. HLA-tetramers - A2 Frequency Phenotype Function A2 Transgenic mouse vaccine model (2/3 months : begin mid 2006) Test single peptides or multi- peptides as vaccine Challenge with BCG/Mtb Examine efficacy Tetramers CD8 expansion Bacterial burden

Papers planned: 1.A2/A3/B7 screening peptide responses: proliferation of CD8 T cells cytokine selected peptides-MHC restriction and functional studies 2.A2 peptide/Transgenic mice: Tetramer studies (frequency, phenotype) Vaccine efficacy 3.CD8 repertoire of BCG vaccinated individuals: cross-sectional/retrospective study

Acknowledgements Prof. Dr. Tom Ottenhoff Tuberculosis group Immunohematology and Blood Transfusion Leiden University Medical Center Leiden, Netherlands Proliferation assays, FACS analysis and IFN-g-ELISA Leucosep Isolation of PBMC Ole Lund, Ph.D.Associate Prof. Immunological bioinformatic group CBS-BioCentrum, DTU Techinical University of Denmark In silico peptide prediction, NetCTL Fatima Kazi Pascale van Weeren And the rest of the Ottenhoff’s group Michel Klein Tom Søren Buus, MD, Ph.D Prof. IMMI, University of Copenhagen MHC binding Ugur Sahin Ganymed Genetic library